Method of cell growth inhibition with agnoprotein

ABSTRACT

The growth of normal and abnormally proliferating cells can be inhibited by the introduction of agnoprotein, or biologically active fragments or derivatives of agnoprotein, into the cell in the absence of any other polyoma virus protein or viral replication.

REFERENCE TO GOVERNMENT GRANT

The invention described herein was supported in part by NIDN/NIH grantnos. P01 NS30916 and P01 NS36466. The U.S. government has certain rightsin this invention.

FIELD OF THE INVENTION

This invention relates to methods of inhibiting cell growth with polyomavirus agnoproteins, especially with agnoprotein from the humanneurotropic polyoma virus JCV.

BACKGROUND OF THE INVENTION

The polyoma viruses (Polyomaviridae) are DNA viruses which infect avariety of species, including man. There are two polyoma viruses thatcause disease in humans; BK virus (isolated from an immunosuppressedkidney transplant patient) and the human neurotropic polyomavirus JCV.Other polyoma viruses include the simian polyoma virus SV40, and themurine and avian polyoma viruses.

JCV is an established etiologic agent of progressive multifocalleukoencephalopathy, a fatal demyelinating disease of humans. Alwine J C(1982), J. Virol. 42, 798-803. Recent studies point to the associationof JCV with several human cancers, including tumors of neural origin.See, e.g., Del Valle L et al., (2001a) Cancer Res 61: 4287-4293;Caldarelli-Stefano R et al., (2000) Human Pathol. 31: 394-395; andKhalili K et al. (1999), Lancet 353: 1152-1153.

As with other polyomaviruses, the genome of JCV is comprised of adouble-stranded circular DNA that contains three functional regions.These functional regions are the early and late coding genomes, and thenon-coding regulatory sequence. Frisque R J et al. (1984), J. Virol. 51:458-469. The early genome is responsible for expression of the viralregulatory protein T-antigen. The late genome is expressed after DNAreplication and results in the accumulation of the capsid proteins VP1,VP2, and VP3. In addition, the leader sequences of the late genometranscripts encompass an open reading frame encoding the agnoprotein.

During the 1980's, several laboratories studied polyomavirusagnoprotein. In particular the biological function of agnoprotein inSV40-infected monkey kidney cells was investigated. See, e.g., Alwine JC (1982) supra and Khalili K (1988), Proc. Natl Acad. Sci. USA 85:354-358. These studies show that SV40 agnoprotein is important for thelate events of the viral lytic cycle. In more recent studies, JCVagnoprotein was shown to interact with early T-antigen. See, e.g., SafakM et al. (2001), J. Virol. 75: 1476-1486. These studies suggest thatT-antigen activity in viral gene transcription and DNA replication maybe dictated, at least in part, by the interaction of T-antigen withagnoprotein. Thus, agnoprotein appears to have an integral function inpolyoma viral replication.

As mentioned above, JCV infection has been linked to various tumors ofcentral nervous system (CNS) origin, including medullablastoma,glioblastoma, and others. Del Valle L et al., (2001a), supra; Khalili K(1999), supra. Examination of T-antigen expression in the CNS tumortissue revealed that not all tumor cells express T-antigen. Evaluationof these tumors for other viral proteins showed a substantial level ofagnoprotein in tumors containing the JCV genome. DeValle L et al.(2002), J. Nat. Cancer Inst. 94(4): 267-273.

The importance of agnogene expression in brain tumor cells is unknown.One hypothesis holds that interactions of T-antigen and agnoprotein witheach other, and with endogenous cellular proteins, may modulate thegrowth rate of tumor cells. Nevertheless, it appears from the studiesdiscussed above that JCV agnoprotein is involved in the development andgrowth of some CNS neoplasms.

SUMMARY OF THE INVENTION

Surprisingly, it has now been found that polyoma virus agnoproteinsinhibit the growth of both normal and abnormally proliferating cells inthe absence of other polyoma viral proteins.

The invention thus provides a method of inhibiting cell growthcomprising introducing into a cell an effective amount of one or moreagnoproteins, or one or more biologically active fragments orderivatives of agnoprotein, such that growth of the cell is inhibited.

The invention also provides a method of treating a subject having acancer or a non-cancerous proliferative disorder, comprisingadministering to the subject an effective amount of one or moreagnoproteins, or one or more biologically active fragments orderivatives of agnoprotein, such that growth of cells of the cancer ornon-cancerous proliferative disorder is inhibited.

The invention also provides a pharmaceutical composition for treating asubject having a cancer or a non-cancerous proliferative disorder,comprising agnoprotein, or a biologically active fragment or derivativeof agnoprotein, and a pharmaceutically acceptable carrier.

The invention further provides a pharmaceutical composition for treatinga subject having a cancer or a non-cancerous proliferative disorder,comprising a nucleic acid sequence encoding agnoprotein, or abiologically active fragment or derivative of agnoprotein, and apharmaceutically acceptable carrier.

Amino Acid Abbreviations

The nomenclature used to describe the peptide compounds of the presentinvention follows the conventional practice wherein the amino group ispresented to the left and the carboxy group to the right of each aminoacid residue. In the formulae representing selected specific embodimentsof the present invention, the amino-and carboxy-terminal groups,although not specifically shown, will be understood to be in the formthey would assume at physiologic pH values, unless otherwise specified.In the amino acid structure formulae, each residue is generallyrepresented by a one-letter or three-letter designation, correspondingto the trivial name of the amino acid, in accordance with the followingschedule:

A Alanine Ala C Cysteine Cys D Aspartic Acid Asp E Glutamic Acid Glu FPhenylalanine Phe G Glycine Gly H Histidine His I Isoleucine Ile KLysine Lys L Leucine Leu M Methionine Met N Asparagine Asn P Proline ProQ Glutamine Gln R Arginine Arg S Serine Ser T Threonine Thr V Valine ValW Tryptophan Trp Y Tyrosine Tyr

Definitions

The expression “amino acid” as used herein is meant to include bothnatural and synthetic amino acids, and both D and L amino acids.“Standard amino acid” means any of the twenty standard L-amino acidscommonly found in naturally occurring peptides. “Nonstandard amino acid”means any amino acid, other than the standard amino acids, regardless ofwhether it is prepared synthetically or derived from a natural source.As used herein, “synthetic amino acid” also encompasses chemicallymodified amino acids, including but not limited to salts, amino acidderivatives (such as amides), and substitutions. Amino acids containedwithin the peptides of the present invention, and particularly at thecarboxy- or amino-terminus, can be modified by methylation, amidation,acetylation or substitution with other chemical groups which can changethe peptide's circulating half life without adversely affecting theirbiological activity. Additionally, a disulfide linkage can be present orabsent in the peptides of the invention.

Amino acids have the following general structure:

Amino acids are classified into seven groups on the basis of the sidechain R: (1) aliphatic side chains, (2) side chains containing ahydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) sidechains containing an acidic or amide group, (5) side chains containing abasic group, (6) side chains containing an aromatic ring, and (7)proline, an imino acid in which the side chain is fused to the aminogroup.

As used herein, “agnoprotein” means the approximately 60-75 amino acidprotein produced from the gene in the late leader region of a polyomavirus, which is located just upstream of the VP2 structural gene.Agnoproteins useful in the practice of the present invention are“biologically active” as defined herein, and have at least about 50%sequence identity, preferably at least about 60% sequence identity, forexample 63% or 64% sequence identity, to SEQ ID NO: 1. More preferably,agnoproteins useful in the invention are biologically active as definedherein and have at least about 80% sequence identity, for example 83% or84% sequence identity, with SEQ ID NO: 1. Particularly preferredagnoproteins are biologically active as defined herein and have 90%, 95%or 98% sequence identity with SEQ ID NO: 1.

“Biologically active” with respect to agnoprotein, or fragments orderivatives of agnoprotein, means the ability of the compound to inhibitthe growth of NIH-3T3 cells according to the in vitro cell growth assaysgiven in Example 2 below. Agnoproteins useful in the practice of thepresent invention specifically include agnoproteins from JCV, BK andSV40 polyoma virus strains.

“Isolated” means altered or removed from the natural state through theactions of a human being. For example, a nucleic acid sequence or apeptide naturally present in a living animal is not “isolated,” but thesame nucleic acid or peptide partially or completely separated from thecoexisting materials of its natural state is “isolated.” An isolatednucleic acid sequence or protein can exist in substantially purifiedform, or can exist in a non-native environment such as, for example, ahost cell.

As used herein, “protecting group” with respect to a terminal aminogroup of a peptide means any of the various amino-terminal protectinggroups traditionally employed in peptide synthesis. Such protectinggroups include, for example, acyl protecting groups such as formyl,acetyl, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl;aromatic urethane protecting groups such as benzyloxycarbonyl; andaliphatic urethane protecting groups, for example, tert-butoxycarbonylor adamantyloxycarbonyl. See Gross and Mienhofer, eds., The Peptides,vol. 3, pp. 3-88 (Academic Press, New York, 1981) for suitableprotecting groups.

As used herein, “protecting group” with respect to a terminal carboxygroup of a peptide means any of various carboxyl-terminal protectinggroups traditionally employed in peptide synthesis. Such protectinggroups include, for example, tert-butyl, benzyl or other acceptablegroups linked to the terminal carboxyl group through an ester or etherbond.

“Derivative” includes any naturally occurring or purposefully generatedagnoprotein which is characterized by single or multiple amino acidsubstitutions, deletions, additions, or replacements. Such derivativesinclude (a) derivatives in which one or more amino acid residues ofagnoprotein are substituted with conservative or non-conservative aminoacids; (b) derivatives in which one or more amino acids are added; (c)derivatives in which one or more of the amino acids include asubstituent group; (d) derivatives in which agnoprotein or a portionthereof is fused to another peptide (e.g., serum albumin or proteintransduction domain); (e) derivatives in which one or more nonstandardamino acid residues (i.e., those other than the 20 standard L-aminoacids found in naturally occurring proteins) are incorporated orsubstituted into the agnoprotein sequence; and (f) derivatives in whichone or more nonamino acid linking groups are incorporated into orreplace a portion of agnoprotein.

“Peptide” and “protein” are used interchangeably, and refer to acompound comprised of at least two amino acid residues covalently linkedby peptide bonds or modified peptide bonds (e.g., peptide isosteres). Nolimitation is placed on the maximum number of amino acids which cancomprise a protein or peptide. The amino acids comprising the peptidesor proteins described herein and in the appended claims are understoodto be either D or L amino acids with L amino acids being preferred. Theamino acid comprising the peptides or proteins described herein can alsobe modified either by natural processes, such as posttranslationalprocessing, or by chemical modification techniques which are well knownin the art. Modifications can occur anywhere in a peptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. It is understood that the same type of modification can bepresent in the same or varying degrees at several sites in a givenpeptide. Also, a given peptide can contain many types of modifications.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cystine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination. See, for instance,PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton,W. H. Freeman and Company, New York, 1993 and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York, 1983; Seifter et al., “Analysisfor protein modifications and nonprotein cofactors”, Meth Enzymol (1990)182:626-646 and Rattan et al., “Protein Synthesis: PosttranslationalModifications and Aging”, Ann NY Acad Sci (1992) 663:48-62.

“Variant” as the term is used herein, is a nucleic acid sequence orpeptide that differs from a reference nucleic acid sequence or peptiderespectively, but retains essential properties of the referencemolecule. Changes in the sequence of a nucleic acid variant can notalter the amino acid sequence of a peptide encoded by the referencenucleic acid, or can result in amino acid substitutions, additions,deletions, fusions and truncations. Changes in the sequence of peptidevariants are typically limited or conservative, so that the sequences ofthe reference peptide and the variant are closely similar overall and,in many regions, identical. A variant and reference peptide can differin amino acid sequence by one or more substitutions, additions,deletions in any combination. A variant of a nucleic acid or peptide canbe a naturally occurring such as an allelic variant, or it can be avariant that is not known to occur naturally. Non-naturally occurringvariants of nucleic acids and peptides can be made by mutagenesistechniques or by direct synthesis.

As used herein, “sequence identity” with respect to a reference peptidecan be computed by using the BLASTP and TBLASTN programs which employthe BLAST (basic local alignment search tool) 2.0.14 algorithm; BLASTPand 20 TBLASTN settings to be used in such computations are indicated inTable 1 below. Amino acid sequence identity is reported under“Identities” by the BLASTP and TBLASTN programs. Techniques forcomputing amino acid sequence identity are well known to those skilledin the art, and the use of the BLAST algorithm is described in Altschulet al. (1990), J. Mol. Biol. 215: 403-10 and Altschul et al. (1997),Nucleic Acids Res. 25:3389-3402, the disclosures of which are hereinincorporated by reference in their entirety.

TABLE 1 Settings to be used for the computation of amino acid sequenceidentity with BLASTP and TBLASTN programs utilizing the BLAST 2.0.14algorithm. Expect Value 10 Filter Low complexity filtering using SEGprogram* Substitution Matrix BLOSUM62 Gap existence cost 11 Per residuegap cost 1 Lambda ratio 0.85 Word size 3 *The SEG program is describedby Wootton and Federhen (1993), Comput. Chem. 17: 149-163.

“Sequence identity” with respect to a reference nucleic acid sequencecan be determined by comparing the sequence identity of two sequences,for example by physical/chemical methods (i.e., hybridization) or bysequence alignment via computer algorithm. Suitable nucleic acidhybridization conditions to determine if a nucleic acid sequencepossesses the required sequence identity to a reference nucleic acidsequence are: 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at50° C. with washing in 2× standard saline citrate (SSC), 0.1% SDS at 50°C.; preferably in 7% (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. withwashing in 1×SSC, 0.1% SDS at 50° C. Suitable computer algorithms todetermine substantial similarity between two nucleic acid sequencesinclude, but are not limited to: GCS program package (Devereux et al.(1984), Nucl. Acids Res. 12: 387), and the BLASTN or FASTA programs(Altschul et al. (1990), J. Mol. Biol. 215: 403). The default settingsprovided with these programs are adequate for determining substantialsimilarity of nucleic acid sequences for purposes of the presentinvention.

“Substantially purified” refers to a peptide or nucleic acid sequencewhich is substantially homogenous in character due to the removal ofother compounds (e.g., other peptides, nucleic acids, carbohydrates,lipids) or other cells originally present. “Substantially purified” isnot meant to exclude artificial or synthetic mixtures with othercompounds, or the presence of impurities which do not interfere withbiological activity, and which can be present, for example, due toincomplete purification, addition of stabilizers, or formulation into apharmaceutically acceptable preparation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is Western blot analysis of protein extracts from U-87MG cellstransfected with a plasmid expressing agnoprotein. Lane 1 representsapproximately 50 μg of total protein extract from untransfected cells.Lanes 2-5 represent, respectively, 50 μg of total protein extract fromcells transfected with pCMV-agnoprotein at 19, 24, 43, and 49 hoursafter transfection. Lanes 6 and 7 represent extracts from uninfected andJCV-infected human astrocytes, respectively. The position of theagnoprotein is shown by an arrow. The asterisk shows the position of anon-specific band. FIGS. 1B and 1C are photomicrographs showing U-87MGcells transfected with a plasmid expressing agnoprotein which were fixedand reacted, respectively, with preimmune or anti-agnoprotein antibodiesat 24 hrs. post-transfection (original magnification 400×).

FIG. 2A is a plot comparing proliferation of NIH 3T3 cells in theabsence and presence of agnoprotein expression. Four independent sampleswere tested after 24, 48, and 72 hours for cell proliferation by directcounting of the total number of cells. All error is represented in SEM.FIGS. 2B and 2C are, respectively, fluorescence-activated cell sorting(FACS) analyses of NIH 3T3 cells stably expressing YFP orYFP-agnoprotein at 0, 16, 21, 25 and 29 hours after serum stimulation.The percentage of cells in G1, S, and G2/M is indicated for each timepoint. Histograms are representative of three experiments and theaverage values are presented.

FIGS. 3A and 3B are, respectively, Western blot analyses of cyclin Aexpression in synchronized NIH 3T3 cells expressing YFP orYFP-agnoprotein at 0, 4, 16, 21 and 29 hours after serum stimulation. 50μg total protein extract was analyzed for each cell type. As a controlfor protein loading in the gels, the levels of Grb2 are shown in thebottom panels. FIG. 3C is a plot showing associated kinase activity ofcyclin A in 220 μg of protein extracted from YFP and YFP-agnoproteinproducing NIH 3T3 cells at 0, 4, 16, 21 and 29 hours after serumstimulation. Kinase activity is expressed as counts-per-minute (cpm) onthe Y axis, and the time after serum stimulation is given in hours onthe X axis. The kinase assay was repeated two times for each sample,giving an interassay standard deviation within 10% after normalizationfor protein amount.

FIGS. 4A and 4B are, respectively, Western blot analyses of cyclin Bexpression in synchronized NIH 3T3 cells expressing YFP orYFP-agnoprotein at 0, 4, 16, 21 and 29 hours after serum stimulation. 50μg total protein extract was analyzed for each cell type. As a controlfor protein loading in the gels, the levels of Grb2 are shown in thebottom panels. FIG. 4C is a plot showing associated kinase activity ofcyclin B in 220 μg of protein extracted from YFP and YFP-agnoproteinproducing NIH 3T3 cells at 0, 4, 16, 21 and 29 hours after serumstimulation. Kinase activity is expressed as counts-per-minute (cpm) onthe Y axis, and the time after serum stimulation is given in hours onthe X axis. Kinase activity is expressed as the average of twoexperiments after normalization for protein levels.

FIGS. 5A-C are, respectively, Western blot analyses of approximately 50μg of total protein extracted from NIH 3T3 (“control”) and NIH3T3-agnoprotein (“Agnoprotein”) producing cell lines using antibodiesthat recognize p27, p21 and β-actin. FIG. 5D is plot of luciferaseactivity in Saos-2 cells transfected with a p21-luciferase reporter genealone, or with plasmids expressing p53 (“p53 +/−”) or agnoprotein(“Agnoprotein+/−”).

FIG. 6A is a Western blot analysis of 50 μg protein extracted fromcontrol and agnoprotein-producing NIH 3T3 cells using anti-p53 antibodyFIG. 6B is an autoradiograph of agnoprotein that was immobilized onglutathione-Sepharose beads, incubated with in vitro translated[³⁵S]-methionine-labeled p53 and resolved by SDS-PAGE. One-tenth of theinput p53 used in each reaction was loaded as a migrating control inlane 1. FIG. 6C is an autoradiograph of p53 fused to GST beads mixedwith in vitro translated [³⁵S]-labeled agnoprotein and resolved bySDS-PAGE. One-tenth of the of the input from the in vitro translationreaction was loaded as a migrating control in lane 1. FIG. 6D is aWestern blot analysis of immunoprecipitates from 150 μg proteinextracted from control NIH 3T3 and agnoprotein-producing NIH 3T3 cells.The position of the fusion agnoprotein is shown by an arrow. Theasterisk indicates a non-specific band seen in both control andexperimental cell extracts. FIG. 6E is a Western blot analysis ofimmnunoprecipitates from approximately 250 μg protein extracted fromcontrol cells expressing YFP or cells expressing GFP-Agnoprotein wereanalyzed by GST pull-down assay using GST or GST-p53 as indicated. Lanes1 and 2 represent an analysis of 50 μg protein extract from each celltype which was used in the binding experiment. The arrow indicates theposition of YFP-Agnoprotein. FIG. 6F is an autoradiograph of in vitro[³⁵S]-methionine-labeled p53 protein analyzed by GST pull-down assayusing various regions of the agnoprotein as indicated, which were fusedto GST. The position of the labeled p53 is indicated by the arrow.

DETAILED DESCRIPTION OF THE INVENTION

Introduction of agnoprotein into a cell, in the absence of other polyomaviral proteins, inhibits growth of the cell. Without wishing to be boundby any particular theory, it appears that agnoprotein deregulates cellgrowth by delaying progression of cells through the various phases ofthe cell cycle. The growth of both normally and abnormally proliferatingcells can be inhibited with agnoprotein.

Agnoprotein from any species and strain of polyoma virus can be used inthe practice of the present invention. Preferred agnoproteins are thosederived from JCV, in particular from the Mad-1 strain of JCV. Theagnoprotein from JCV strain Mad1 is a 71 amino acid protein with thefollowing primary sequence:

(SEQ ID NO:1)     MVLRQLSRKASVKVSKTWSGTKKRAQRILIFLLEFLLDFCTGEDSVDGKKRQRHSGLTEQTYSALPEPKAT

The nucleic acid sequence encoding SEQ ID NO: 1 is given in SEQ ID NO:2.

Other JCV agnoproteins known in the art include those disclosed inGenBank records Accession No. AF187236 (SEQ ID NO: 3), Accession No.AF281599 (SEQ ID NO: 4), Accession No. AF187234 (SEQ ID NO: 5),Accession No. AF295737 (SEQ ID NO: 6) and Accession No. AF295739 (SEQ IDNO: 7), the entire disclosures of which are herein in incorporated byreference. The nucleic acid sequences encoding these agnoproteins aregiven, respectively, in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQID NO: 11, and SEQ ID NO: 12.

See also Cubitt C L et al. (2001), J. Neurovirol. 7: 339-344, thedisclosure of which is herein incorporated by reference, in whichpredicted agnoprotein amino acid sequences for 100 JCV strains wereanalyzed to produce the following agnoprotein consensus sequence:

(SEQ ID NO:13)       M-V-L-R-Q-L-S-R-K-A-S-V-K-V-S-K-T-W-S-G-T-K-K-R-A-Q-R-I-L-I-F-L-L-E-F-L-L-D-F-C-T-G-E-D-X₁-V-D-G-K-K-R-Q-X₂-H-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-A-L-P-E-P-K-A-X₁₂

wherein

-   -   X₁ is serine or arginine;    -   X₂ is lysine or arginine;    -   X₃ is serine or arginine;    -   X₄ is glycine or no amino acid;    -   X₅ is leucine or no amino acid;    -   X₆ is threonine or no amino acid;    -   X₇ is glutamine, glutamic acid, or no amino acid;    -   X₈ is glutamine or no amino acid;    -   X₉ is threonine, arginine, lysine or no amino acid;    -   X₁₀ is tyrosine or no amino acid;    -   X₁₁ is serine or glycine; and    -   X₁₂ is threonine or lysine.        A protein comprising SEQ ID NO: 13 is considered an agnoprotein,        and can be used to inhibit growth of cells according to the        present invention.

Agnoprotein from human BK polyoma virus strains, or from SV40 polyomavirus strains, are also known. Examples of BK virus agnoproteins arefound in GenBank record Accession Nos. M23122 (SEQ ID NO: 14) and D00678(SEQ ID NO: 15), and a partial BK virus agnoprotein sequence is found inAccession No. AF442903 (SEQ ID NO: 16), the disclosures of which areherein incorporated by reference. An example of SV40 agnoprotein isfound in GenBank record Accession No. M99359 (SEQ ID NO: 17), thedisclosure of which is herein incorporated by reference.

Nucleic acid sequences encoding the BK virus and SV40 agnoproteinsdescribed above are given in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, and SEQ ID NO: 21 respectively.

Agnoproteins can be isolated from mammalian cells infected with polyomaviruses according to known techniques. Agnoproteins can also be producedsynthetically by any known means, including synthesis by biologicalsystems and by chemical methods.

Biological synthesis of peptides is well known in the art, and includesthe transcription and translation of a naturally-occurring or syntheticgene encoding agnoprotein sequences. These nucleic acids can besubcloned into an appropriate plasmid expression vector for propagationand expression in an appropriate host. For example, techniques forcloning and expression of the agnoprotein of SEQ ID NO: 1 are describedin the Examples below.

Other techniques used to construct nucleic acid sequences and plasmidexpression vectors, transfect host cells, and express a nucleic acidsequence of interest are widely practiced in the art, and practitionersof ordinary skill are familiar with the standard resource materialswhich describe specific conditions and procedures. For example, generalmethods for the cloning and expression of recombinant molecules aredescribed in Sambrook et al., Molecular Cloning, Cold Spring HarborLaboratories, 1982; and in Ausubel, Current Protocols in MolecularBiology, Wiley and Sons, 1987, the disclosures of which are incorporatedherein by reference.

Agnoprotein produced from an expression vector can be obtained from thehost cell by cell lysis, or by using heterologous signal sequences fusedto the expressed protein which cause secretion of the protein into thesurrounding medium. Preferably, the signal sequence is designed so thatit can be removed by chemical or enzymatic cleavage, as is known in theart. The agnoprotein thus produced can then be purified in a mannersimilar to that utilized for isolation of agnoprotein from isolated frommammalian cells infected with polyoma viruses.

Chemical peptide synthesis techniques suitable for directly synthesizingagnoprotein, including manual and automated techniques, are alsowell-known to those of ordinary skill in the art. For example,agnoprotein can be synthesized de novo using conventional solid phasesynthesis methods. In such methods, the peptide chain is prepared by aseries of coupling reactions in which the constituent amino acids areadded to the growing peptide chain in the desired sequence. The use ofvarious N-protecting groups, e.g., the carbobenzyloxy group or thet-butyloxycarbonyl group; various coupling reagents e.g.,dicyclohexylcarbodiimide or carbonyldimidazole; various active esters,e.g., esters of N-hydroxyphthalimide or N-hydroxy-succinimide; and thevarious cleavage reagents, e.g., trifluoroactetic acid (TFA), HCl indioxane, boron tris-(trifluoracetate) and cyanogen bromide; and reactionin solution with isolation and purification of intermediates are methodswell-known to those of ordinary skill in the art.

A preferred chemical peptide synthesis method follows conventionalMerrifield solid phase procedures well known to those skilled in theart. Additional information about solid phase synthesis procedures canbe had by reference to Steward and Young, Solid Phase Peptide Synthesis,W.H. Freeman & Co., San Francisco, 1969; the review chapter byMerrifield in Advances in Enzymology 32:221-296, (Nold FF, ed.),Interscience Publishers, New York, 1969; and Erickson and Merrifield(1990), The Proteins 2:61-64, the entire disclosures of which areincorporated herein by reference. Crude peptide preparations resultingfrom solid phase syntheses can be purified by methods well known in theart, such as preparative HPLC. The amino-terminus can be protectedaccording to the methods described for example by Yang et al., FEBSLett. 272:61-64 (1990), the entire disclosure of which is hereinincorporated by reference.

Agnoproteins for use in the present invention can also comprise a labela (e.g., substances which are magnetic resonance active; radiodense;fluorescent; radioactive; detectable by ultrasound; detectable byvisible, infrared or ultraviolet light) so that the agnoprotein can bedetected. Suitable labels include, for example, fluoresceinisothiocyanate (FITC); peptide chromophores such as phycoerythrin orphycocyanin and the like; bioluminescent peptides such as theluciferases originating from Photinus pyrali; fluorescent proteinsoriginating from Renilla reniformi; and radionuclides such as ³²P, ³³P,35S, I¹²⁵ or ¹²³I. For example, the label can comprise an NH₂-terminalfluorescein isothiocyanate (FITC)-Gly-Gly-Gly-Gly motif that isconjugated to a protein transduction domain.

Methods of modifying peptide sequences such as agnoprotein with labelsare well known to those skilled in the art. For example, methods ofconjugating fluorescent compounds such as fluorescein isothiocyanate toshort peptides are described in Danen et al., Exp. Cell Res., 238:188-86(1998), the entire disclosure of which is incorporated herein byreference.

Biologically active fragments of agnoprotein can also be used in thepresent methods. Biologically active agnoprotein fragments according tothe invention can be obtained, for example, by chemical or enzymaticfragmentation of larger natural or synthetic agnoproteins, or bybiological or chemical syntheses as described above.

Biologically active derivatives of agnoprotein can also be used in thepresent methods. The techniques for obtaining such derivatives are knownto persons having ordinary skill in the art and include, for example,standard recombinant nucleic acid techniques, solid phase peptidesynthesis techniques and chemical synthetic techniques as describedabove. Linking groups can also be used to join or replace portions ofagnoprotein and other peptides. Linking groups include, for example,cyclic compounds capable of connecting an amino-terminal portion and acarboxyl terminal portion of agnoprotein. Techniques for generatingderivatives are also described in U.S. Pat. No. 6,030,942 the entiredisclosure of which is herein incorporated by reference (derivatives aredesignated “peptoids” in the U.S. Pat. No. 6,030,942). Agnoproteinderivatives can also comprise labels such as are described above.

Agnoprotein derivatives also include fusion peptides in which a portionof the fusion peptide has a substantially similar amino acid sequence toagnoprotein. Such fusion peptides can be generated by techniqueswell-known in the art, for example by subcloning nucleic acid sequencesencoding an agnoprotein and a heterologous peptide sequence into thesame expression vector, such that the agnoprotein and the heterologoussequence are expressed together in the same protein. The heterologoussequence can comprise a peptide leader sequence that directs entry ofthe expressed protein into a cell. Such leader sequences include“protein transduction domains” or “PTDs”, which are discussed in moredetail below.

A preferred agnoprotein derivative comprises the JCV agnoproteindescribed in Jobes D V et al. (1999), J. Human Virol. 2(6): 350-358, thedisclosure of which is herein incorporated by reference, which has a7-amino acid deletion in the C-terminal region (SEQ ID NO: 22). Thenucleic acid sequence encoding SEQ ID NO: 22 is given in SEQ ID NO: 23.

The agnoproteins and biologically active fragments and derivatives ofagnoproteins described above are also referred to hereunder as“compounds of the invention.”

The compounds of the invention can be modified to enhance their entryinto cells. For example, the compounds of the invention can beencapsulated in a liposome prior to being administered. The encapsulatedcompounds are delivered directly into the abnormally proliferating cellsby fusion of the liposome to the cell membrane. Reagents and techniquesfor encapsulating the present compounds in liposomes are well-known inthe art, and include, for example, the ProVectin™ Protein DeliveryReagent from Imgenex.

In a preferred embodiment, the compounds of the invention are modifiedby associating the compounds with a peptide leader sequence known as a“protein transduction domain” or “PTD.” These sequences direct entry ofthe compound into abnormally proliferating cells by a process known as“protein transduction.” See Schwarze et al. (1999), Science 285:1569-1572.

PTDs are well-known in the art, and can comprise any of the known PTDsequences including, for example, arginine-rich sequences such as apeptide of nine to eleven arginine residues optionally in combinationwith one to two lysines or glutamines as described in Guis et al.(1999), Cancer Res. 59: 2577-2580, the disclosure of which is hereinincorporated by reference. Preferred are sequences of eleven arginineresidues or the NH₂-terminal 11-amino acid protein transduction domainfrom the human immunodeficiency virus TAT protein (SEQ ID NO: 24). Othersuitable leader sequences include, but are not limited to, otherarginine-rich sequences; e.g., 9 to 10 arginines, or six or morearginines in combination with one or more lysines or glutamines. Suchleader sequences are known in the art; see, e.g., Guis et al. (1999),supra. Preferably, the PTD is designed so that it is cleaved from thecompound upon entry into the cell. A PTD can be located anywhere on theagnoprotein, or fragment or derivative of agnoprotein, that does notdisrupt the compound's biological activity, and is preferably located atthe N-terminal end.

Kits and methods for constructing fusion proteins comprising a proteinof interest (e.g., agnoprotein) and a PTD are known in the art; forexample the TransVector™ system (Q-BIOgene), which employs a 16 aminoacid peptide called “Penetratin™” corresponding to the Drosophilaantennapedia DNA-binding domain; and the Voyager system (Invitrogen LifeTechnologies), which uses the 38 kDa VP22 protein from Herpes SimplexVirus-1.

Agnoprotein, or biologically active fragments or derivatives ofagnoprotein, can inhibit proliferation of normal and abnormallyproliferating cells. Abnormally proliferating cells include cells fromcancer types of diverse histologic subtype and origin, such as thoselisted and described in the National Cancer Institute's “CancerNet,”which is herein incorporated by reference in its entirety.

For example, the compounds of the invention can be used to inhibit theproliferation of primary or metastatic tumor or neoplastic cells fromcancers of at least the following histologic subtypes: sarcoma (cancersof the connective and other tissue of mesodermal origin); melanoma(cancers deriving from pigmented melanocytes); carcinoma (cancers ofepithelial origin); adenocarcinoma (cancers of glandular epithelialorigin); cancers of neural origin (glioma/glioblastoma and astrocytoma);and hematological neoplasias, such as leukemias and lymphomas (e.g.,acute lymphoblastic leukemia, chronic lymphocytic leukemia, and chronicmyelocytic leukemia).

The compounds of the invention can also be used to inhibit theproliferation of primary or metastatic tumor or neoplastic cells fromcancers having their origin in at least the following organs or tissues,regardless of histologic subtype: breast; tissues of the male and femaleurogenital system (e.g. ureter, bladder, prostate, testis, ovary,cervix, uterus, vagina); lung; tissues of the gastrointestinal system(e.g., stomach, large and small intestine, colon, rectum); exocrineglands such as the pancreas and adrenals; tissues of the mouth andesophagus; brain and spinal cord; kidney (renal); pancreas;hepatobiliary system (e.g., liver, gall bladder); lymphatic system;smooth and striated muscle; bone and bone marrow; skin; and tissues ofthe eye.

Furthermore, the compounds of the invention can be used to inhibit theproliferation of cells from cancers or tumors in any prognostic stage ofdevelopment, as measured, for example, by the “Overall Stage Groupings”(also called “Roman Numeral”) or the Tumor, Nodes, and Metastases (TNM)staging systems. Appropriate prognostic staging systems and stagedescriptions for a given cancer are known in the art, for example asdescribed in the National Cancer Institute's “CancerNet,” supra.

Agnoprotein, or biologically active fragments or derivatives ofagnoprotein, can also be used to inhibit proliferation of cells fromnon-cancerous proliferative disorders. The non-cancerous proliferativedisorders are characterized by cells which have escaped normal growthcontrols, but are not able to metastasize. Abnormally proliferatingcells in such disorders typically form fibroid growths or benign tumors.

Examples of non-cancerous proliferative disorders include any benignskin lesion or condition involving the uncontrolled growth offibroblasts, hemangiomatosis in newborn; secondary progressive multiplesclerosis; chronic progressive myelodegenerative disease;neurofibromatosis; ganglioneuromatosis; keloid formation; Paget'sDisease of the bone; fibrocystic disease (e.g., of the breast, lungs, oruterus); sarcoidosis; Peronies' and Duputren's fibrosis, cirrhosis,atherosclerosis and vascular restenosis.

Preferably, the compounds of the invention are used to treat a subjecthaving abnormally proliferating cells deriving from a cancer or anon-cancerous proliferative disorder. In treating subjects with suchconditions, one or more compounds of the invention are administered to asubject in an amount effective to inhibit proliferation of abnormallyproliferating cells (the “effective amount”). The subject can be anyanimal, preferably a mammal, particularly preferably a human being.

As used herein, to “inhibit the proliferation of an abnormallyproliferating cell” means to kill the cell, or permanently ortemporarily arrest the growth of the cell.

Inhibition of proliferation can be inferred if the number of abnormallyproliferating cells in the subject remains constant or decreases afteradministration of the present compounds. Inhibition of proliferation canalso be inferred if the absolute number of abnormally proliferatingcells increases, but the rate of growth of a tissue mass decreases. Asused herein, a “tissue mass” is any localized collection of abnormallyproliferating cells in a subject's body; for example a tumor, fibroidbody, restenotic plaque, and the like. The number of abnormallyproliferating cells in a subject's body can be determined by directmeasurement (e.g., calculating the concentration of leukemic cells inthe blood or bone marrow) or by estimation from the size of a tissuemass.

The size of a tissue mass can be ascertained by direct visualobservation or by diagnostic imaging methods such as X-ray, magneticresonance imaging, ultrasound, and scintigriphy. Diagnostic imagingmethods used to ascertain size of a tissue mass can be employed with orwithout contrast agents, as is known in the art. The size of a tissuemass can also be ascertained by physical means, such as palpation of thetissue mass or measurement of the tissue mass with a measuringinstrument such as a caliper.

Agnoprotein, or biologically active fragments or derivatives ofagnoprotein, can be administered to a subject by any technique designedto expose abnormally proliferating cells in the subject's body to thecompounds, such that the compounds are taken up by the cells. Forexample, the compounds of the invention can be administered by anyenteral or parenteral route. Parenteral administration is preferred.

Suitable parenteral administration methods include intravascularadministration (e.g. intravenous bolus injection, intravenous infusion,intra-arterial bolus injection, intra-arterial infusion and catheterinstillation into the vasculature); peri- and intra-tissue injection(e.g. peri-turnoral and intra-tumoral injection); subcutaneous injectionor deposition including subcutaneous infusion (such as by osmoticpumps); and direct application to the abnormally proliferating cells orto tissue comprising the abnormally proliferating cells, for example bya catheter or other placement device. It is preferred that subcutaneousinjections or infusions be given in the area near the abnormallyproliferating cells, particularly if the cells are on or near the skin.

The compounds of the invention can be injected in a single dose or inmultiple doses. Infusion of the compounds of the invention can comprisea single sustained dose over a prolonged period of time or multipleinfusions. Direct injection into tissue comprising the abnormallyproliferating cells is preferred.

An effective amount of the compounds of the invention can be based onthe approximate weight of the tissue mass to be treated. The approximateweight of a tissue mass can be determined by calculating the approximatevolume of the tissue mass, wherein one cubic centimeter of tissue massvolume is roughly equivalent to one gram. Where more than one compoundof the invention is administered, the effective amount represents thecumulative total of the administered compounds.

An effective amount of the compounds of the invention based on theweight of a tissue mass can be at least about 10 μg compound/gram oftissue mass, and is preferably between about 10-1000 μg compound/gram oftissue mass. More preferably, the effective amount is at least about 60μg compound/gram of tissue mass. Particularly preferably, the effectiveamount is at least about 100 μg compound/gram of tissue mass. It ispreferred that effective amounts based on the weight of the tissue massbe injected directly into the tissue mass.

An effective amount of the compounds of the invention can also be basedon the approximate or estimated body weight of the subject to betreated. Preferably, such effective amounts are administeredsystemically; e.g. by intravascular injections and infusions,subcutaneous depositions or infusions, or intramuscular orintraperitoneal administrations. Where more than one compound of theinvention is administered, the effective amount represents thecumulative total of the administered compounds.

For example, an effective amount of the compounds of the inventionadministered by single intravascular injection in humans (assuming a 60kg subject) can range from about 5-3000 μg compound/kg of body weight,is preferably between about 700-1000 μg compound/kg of body weight, andis more preferably greater than about 1000 μg compound/kg of bodyweight.

An effective amount of the compounds of the invention used for multipleintravascular injections can be the same or lower than that used forsingle intravascular injections. For example, an effective amount formultiple intravascular injection in humans is preferably greater thanabout 250 μg compound/kg body weight, and is more preferably greaterthan about 500 μg compound/kg body weight.

An effective amount of the compounds of the invention administered bysingle sustained intravascular infusion can be the same as that used forsingle and multiple intravascular injections, but can also be lower. Forexample, an effective amount for single sustained infusions in humans ispreferably greater than about 90 μg compound/kg body weight, and is morepreferably greater than about 100 μg compound/kg body weight.

An effective amount of the compounds of the invention administered bymultiple sustained intravascular infusions can be the same as that usedfor single and multiple injections and single sustained intravascularinfusion, but can also be lower. For example, an effective amount formultiple sustained intravascular infusions in humans is preferablygreater than about 35 μg/kg, and is more preferably greater than about50 μg/kg.

An effective amount of the compounds of the invention administered bysubcutaneous, intramuscular or intraperitoneal routes can be the same asthat used for intravascular administration, but is preferably betweenabout 200 and 1000 μg compound/kg body weight. More preferably, theeffective amount is greater than 500 μg compound/kg of body weight.

An effective amount of the compounds of the invention can also be basedon the approximate surface area of the subject to be treated. Effectiveamounts based on surface area are typically expressed in terms of μgcompound/square meter of surface area (m²). It is preferred to baseeffective amounts on the surface area of a subject, because betterinter-species comparisons can be made. Also, effective amounts based onsurface area allow amounts to be determined for human adults andchildren without further adjustment. The assumptions underlying theinter-species and adult to child conversion of effective amounts basedon surface area are found in E J Freireich et al., (1966), CancerChemotherapy Reports 50: 219-244, the disclosure of which is hereinincorporated by reference in its entirety.

Table 2 provides approximate surface area-to-weight ratios for variousspecies. The surface area-to-weight ratio can be used to converteffective amounts based on body weight (expressed in μg/kg) to effectiveamounts based on surface area (expressed in μg/m²). The surfacearea-to-weight ratio is also used to calculate the conversion factorsfound in Table 3, which can be used to convert effective amountsexpressed in terms of μg/kg from one species to another.

TABLE 2 Surface Area to Weight Ratios of Various Species* Surface Areato Species Body Weight (kg) Surface Area (m²) Weight Ratio (kg/m²) Mouse0.02 0.0066 3.0 Rat 0.15 0.025 5.9 Monkey 3 0.24 12 Dog 8 0.40 20 Humanchild 20 0.80 25 adult 60 1.6 37 *Adapted from DeVita, VT, “Principlesof Chemotherapy,” pgs. 292-3, in Cancer: Principles and Practice ofOncology, (3rd edit., DeVita VT, Hellman S, and Rosenberg SA, eds.),1989, J. B. Lipincott Co., Phila., PA.

As shown in Table 2, to convert an effective amount based on body weightin μg/kg for any given species to the equivalent effective amount basedon surface area (in μg/m²), multiply the effective amount based on bodyweight by the approximate surface area to weight ratio. For example, inthe adult human 100 μg/kg is equivalent to 100 μg/kg×37 kg/m²=3700μg/m².

Table 3 gives approximate factors for converting effective amountsexpressed in terms of μg/kg from one species to an equivalent surfacearea effective amount expressed in the same units (μg/kg) for anotherspecies. For example, given a dose of 50 μg/kg in the mouse, theappropriate dose in man (assuming equivalency on the basis of μg/m²) is50 μg/kg× 1/12=4.1 μg/kg. For the present invention, equivalency on thebasis of μg/m² is assumed.

TABLE 3 Equivalent Surface Area Dosage Conversion Factors* Mouse RatMonkey Dog Man (20 g) (150 g) (3 kg) (8 kg) (60 kg) Mouse 1 ½ ¼ ⅙ 1/12Rat 2 1 ½ ¼ 1/7 Monkey 4 2 1 ⅗ ⅓ Dog 6 4 5/3 1 ½ Man 12  7 3 2 1*Adapted from DeVita, VT, “Principles of Chemotherapy,” pgs. 292-3, inCancer: Principles and Practice of Oncology, (3^(rd) edit., DeVita V T,Hellman S, and Rosenberg S A, eds.), 1989, J. B. Lipincott Co., Phila.,PA.

An effective amount of the compounds of the invention based on surfacearea is preferably administered systemically, as described above foreffective amounts based on body weight. However, effective amounts basedsurface area can also be administered by peri- or intra-tissue massinjection or by direct application to the tissue mass. Where more thanone compound of the invention is administered, the effective amountrepresents the cumulative total of the administered compounds.

Agnoprotein, and biologically active fragments and derivatives ofagnoprotein, can also be administered to a subject by transfection ofthe abnormally proliferating cells in the subject's body with one ormore nucleic acid sequences encoding a compound of the invention.Preferably, the nucleic acid sequences comprise a plasmid expressionvector. Such plasmids can be generated by recombinant nucleic acid andmolecular cloning techniques well-known in the art, as discussed above.

Transfection methods for eukaryotic cells are well known in the art, andinclude, for example, direct injection of the nucleic acid into thenucleus or pronucleus; electroporation; liposome transfer; receptormediated nucleic acid delivery, bioballistic or particle acceleration;calcium phosphate precipitation, and transfection mediated by viralvectors.

For example, the transfection is performed with a liposomal transfercompound, e.g., DOTAP(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate,Boehringer—Mannheim) or an equivalent, such as LIPOFECTIN. The amount ofnucleic acid used is not critical to the practice of the invention;acceptable results can be achieved with 10 mM nucleic acid/10⁵ cells. Aratio of about 500 nanograms of plasmid vector in 3 micrograms of DOTAPper 10⁵ cells can be used. Other suitable methods for the constructionand propagation of plasmid vectors capable of expressing the presentcompounds, and techniques for transfecting such vectors into eukaryoticcells so that the compounds are expressed, are known in the art. Apreferred transfection method is the calcium phosphate precipitationmethod described in the Examples below.

The present invention also provides pharmaceutical formulations fortreating cancer or non-cancerous proliferative disorders. Thepharmaceutical compositions of the invention comprise one or moreagnoproteins, or one or more biologically active fragments orderivatives of agnoprotein. The pharmaceutical formulations of thepresent invention can also comprise one or more nucleic acids encodingan agnoprotein or a biologically active fragment or derivative ofagnoprotein.

Pharmaceutical formulations of the present invention are characterizedas being at least sterile and pyrogen-free. As used herein,“pharmaceutical formulations” include formulations for human andveterinary use. The present pharmaceutical formulations can be preparedby mixing agnoprotein, biologically active fragments or derivatives ofagnoprotein, or nucleic acids encoding these compounds with aphysiologically acceptable carrier medium to form solutions, suspensionsor dispersions. Preferred physiologically acceptable carrier media arewater or normal saline.

Pharmaceutical formulations of the invention can also compriseconventional pharmaceutical excipients and/or additives. Suitablepharmaceutical excipients include stabilizers, antioxidants, osmolalityadjusting agents, buffers, and pH adjusting agents. Suitable additivesinclude physiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions (e.g., 0.01 to 10 mole percent) of chelants(such as, for example, DTPA or DTPA-bisamide) or calcium chelatecomplexes (as for example calcium DTPA, CaNaDTPA-bisamide), or,optionally, additions (e.g. 1 to 50 mole percent) of calcium or sodiumsalts (for example, calcium chloride, calcium ascorbate, calciumgluconate or calcium lactate). Pharmaceutical formulations of theinvention can be prepared in a manner fully within the skill of the art.

The invention will now be illustrated with the following non-limitingexamples.

EXAMPLES General Experimental Procedures

The following general experimental procedures were used in the Examplesdiscussed below.

Cell Culture and Transfection—Human astrocytoma cell line U-87MG (ATCCcatalog no. HTB-14), human glioblastoma multiforme cell line T98G (ATCCcatalog no. CRL-1960), mouse fibroblast NIH 3T3 cells (ATCC catalog no.CRL-1658), and cell line Saos-2 were maintained in Dulbecco's ModifiedEagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).Transfections were performed on approximately 5-7×10⁵ cells per 60 mmdish using the calcium phosphate precipitation method, according to themethod of Graham FL and van der Eb A J (1973) Virology 52, 456-467, theentire disclosure of which is herein incorporated by reference. At 48hours post-transfection, cells were harvested for protein extractpreparation or were transferred to 100 mm plates containing DMEM plus10% FBS and Geneticin at a concentration of 0.5 mg/ml for the selectionof stably transfected cells.

Plasmids—The plasmids that were used in the various transfections werepCMV-agnoprotein, pCMV-GFP, pGEX-1λT-agnoprotein, pYFP-agnoprotein,pEYFP-N1, pTR(AAV)-Agno, and p21-luciferase.

pCMV-agnoprotein and pGEX-1λT-agnoprotein are described in Safak M etal. (2001) J. Virol. 75, 1476-1486, the entire disclosure of which isherein incorporated by reference.

pEGFP-N1 was obtained from Clontech.

pYFP-agnoprotein was created by PCR amplification of the gene encodingthe agnoprotein from the plasmid pBJC (which contained the Mad-1 strainof JCV) using the following primers containing a BamHI and EcoRI site,respectively:

(SEQ ID NO:25) 5′-ACGTCCAGGATCCATGGTTCTTCGCCAGCTGTCA-3′ (SEQ ID NO:26)5′-ACGTCCAGAATTCCTATGTAGCTTTTGGTTCAGG-3′After gel purification, the amplicon was digested with BamHI and EcoRIand subcloned into the same sites of the pEYFP-N1 vector multiplecloning site (Clontech). All constructs were verified by sequencing. ThepEYFP-N1 vector, which expresses only YFP, was used as a control.

pTR(AAV)-Agno was constructed by amplifying the sequence encoding theagnoprotein with the following primers, which contain NotI sites andcreate a T7 promoter for the amplicon:

5′-TATGCGGCCGCTAATACGACTCACTATAGG-3′ (SEQ ID NO:27)5′-TAGAATAGGGCCCTCTAGATGCATGCTCGA-3′ (SEQ ID NO:28)The amplicon was then digested with NotI and inserted into the NotI siteof the pTR(AAV) vector. All constructs were verified by sequencing.

p21-luciferase was obtained from Dr. B. Sawaya, Center for Neurovirologyand Cancer Biology, Temple University, Philadelphia, Pa.

Protein Extract Preparation—For protein extraction, cells were lysed for20 min on ice in lysis buffer (20 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.5%NP40) containing 10 μg/ml leupeptin, 2 μg/ml aprotinin, 100 μg/ml PMSF,100 μg/ml TPCK; and 10 μg/ml pepstatin. Cell debris was removed bycentrifugation and the supernatant was collected for proteinconcentration by Bradford analysis (Bio-Rad, Hercules, Calif.).

Western Blot Analysis—Approximately 50 μg of the protein extracts wereseparated by SDS-PAGE (12% gel) and analyzed by Western blot using thespecific antibodies set forth in the Examples below. Briefly, proteinsresolved by SDS-PAGE were transferred to Optitran-supportednitrocellulose (Schleicher & Schuell) and blocked in PBS-T (PBS-0.1%Tween 20) containing 10% non-fat dry milk for 45 min. Blots were thenincubated in primary antibody for two hours, washed 3×10 min in PBS-T,incubated with anti-mouse or anti-rabbit secondary antibody conjugatedto horseradish peroxidase for an additional hour, followed by washing3×10 min in PBS-T. Blots were developed with ECL-Plus (AmershamPharmacia, Piscataway, N.J.) and detected by autoradiography.

Immunoprecipitation—150-500 μg of protein extracts were incubated withanti-p53 antibody overnight at 4° C., with rotation. This solution wasthen incubated with 30 μl of pre-washed protein A Sepharose for 2 hoursat 4° C. to precipitate the protein complexes. The precipitatedSepharose A/protein complexes were washed twice in lysis buffer,resuspended in 10 μl of Laemmli sample buffer (62.5 mM Tris-HCl pH 6.8;2% SDS; 25% glycerol; 0.01% Bromophenol Blue) (Biorad), and incubatedfor 15 min at room temperature. Samples were centrifuged, thesupernatant was boiled at 95° C. for 10 min and resolved by SDS-PAGEfollowed by Western blot analysis as above, using anti-agnoproteinantibody.

H1 kinase assay—Approximately 200 μg of protein extract wasimmunoprecipitated with antibodies as specified in the Examples below.Approximately 30 μl of packed protein-A Sepharose beads in lysis bufferwas added to the extracts and incubated at 4° C. for 3 hours. Theimmunoprecipitates were assayed for kinase activity for 30 min at 30°C., in assay buffer containing 5 μCi of [γ³²P]ATP, 5 μg of histone H1, 1mM DTT, 10 mM MgCl₂, and 20 mM Tris (pH 7.4). The reaction was stoppedby the addition of Laemmli sample buffer (BioRad) and analyzed bySDS-PAGE followed by autoradiography.

Cell Proliferation and Flow Cytometry—Cells were plated in 35 mm petridishes at a density of 1×10⁴ cells/plate and maintained in DMEM plus 10%FBS. Cells were collected at various times and cell proliferation wasassessed by determining total cell number by phase contrast microscopy.

For flow cytometry, cells were synchronized by serum starvation for 72hrs. and then stimulated by the addition of growth media containingserum. At various times after serum stimulation, as indicated in theExamples, cells were collected by centrifugation and were resuspended in300 μl of PBS containing 10 μg/ml of propidiun iodide and 250 μg/ml ofRNase A. Samples were incubated at 37° C. for 30 min and kept at 4° C.until analyzed. Cells were analyzed by flow cytometry (i.e.,fluorescence activated cell sorting, or “FACS”) with a Becton Dickinson“FACScan” flow cytometer using the SOBR program.

In Vitro GST Pull-Down Assay—Three microliters of [³⁵S]-labeled in vitrotranslated p53 or agnoprotein were incubated with 5 μg of GST or fusionproteins, GST-agnoprotein or GST-p53 coupled to glutathione Sepharosebeads in 300 μl of LB150 buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 5mM EDTA; 0.1% NP40) for one hour at 4° C., with continuous rocking.After incubation, the beads were precipitated and washed five times withLB150 buffer. The bound proteins were eluted with Laemmli sample buffer(BioRad), heated at 95° C. for 10 min, and resolved by SDS-PAGE. p53 oragnoprotein was detected by autoradiography.

Example 1 Expression of Agnoprotein in Glial Cells and Effect ofAgnoprotein on Cell Cycle Progression

U-87MG cells were transfected with the agnoprotein-expressing plasmidpCMV-agnoprotein, and the level of agnoprotein was determined at 19, 24,43 and 49 hrs. post-transfection. Untransfected U-87MG cells were usedas a control. As shown in FIG. 1A, Western blot analysis of proteinextracts from control cells (lane 1) and transfected cells at thevarious time points (lanes 2-5) showed detectable levels of agnoproteinin transfected cells from 19 hours post-transfection, with accumulationof agnoprotein up to 49 hours post-transfection. FIG. 1A also shows aband corresponding to agnoprotein in normal human astrocytes infectedwith JCV at 15 days post-infection (lane 7), and its absence inuninfected cells (lane 6).

Subcellular localization of agnoprotein in U-87MG cells transfected withpCMV-agnoprotein was also determined. Immunohistochemical examination oftransfected U-87MG cells revealed that incubation with pre-immune seradid not detect agnoprotein (FIG. 1B). However, incubation of transfectedU-87MG cells with rabbit anti-Agno protein (prepared as described inDeValle L et al. (2002), J. Nat. Cancer Inst. 94(4): 267-273, the entiredisclosure of which is herein incorporated by reference) showedcytoplasmic perinuclear accumulation of agnoprotein in the cells (FIG.1C).

To determine the effect of agnoprotein expression on cell cycleprogression, U-87MG cells were synchronized and serum stimulated asabove. At 4 hrs. after serum stimulation, cells were co-transfected withboth pCMV-agnoprotein (which expresses agnoprotein) and pCMV-GFP (whichexpresses green fluorescent protein, or “GFP”). In parallel, controlU-87MG cells were transfected with pCMV-GFP alone. At 23, 28, 47 and 53hrs. post-stimulation, cells were harvested and the percentages of cellsin G1, S, and G2/M were determined by flow cytometry as described above.The time points measured encompassed one complete round of progressionthrough the cell cycle. Cells that expressed GFP were “gated” on theFACS, and the DNA profile of GFP-positive cells from thepCMV-agnoprotein/pCMV-GFP group was compared to that of the controlcells transfected with only pCMV-GFP. The results are presented below inTable 4.

TABLE 4 Cell Cycle Progression of U-87MG Cells Expressing GFP orGFP-Agnoprotein Hrs. Control Experimental Post Serum (% GFP Cells) (%GFP + Agno Cells) Induction G1 S G2/M G1 S G2/M 23 67 17 15 27 21 51 2829 17 53 28 18 53 47 67 13 19 66 14 18 53 32 15 52 62 12 25

For the pCMV-GFP control cell group, nearly 67% of the cells were foundat the G1 stage and only 15% accumulated at the G2/M stage at 23 hrs.after serum stimulation. These values had significantly changed by 28hrs. post-stimulation, as only 29% of the cells were found in the G1phase and more than 53% of the cells had progressed to the G2/M phase.At 47 hrs. post-serum stimulation, 67% of the cells were again found atthe G1 stage, and only a minor fraction of the cells (19%) were detectedat G2/M. As time progressed, more cells departed from the G1 phase andaccumulated in G2/M.

In contrast to the control cells, the majority ofpCMV-agnoprotein/pCMV-GFP cells (51%) were found at the G2/M phase andonly 27% of the cells were detected at G1 stage at 23 hrs.post-stimulation. At 28 hours post-stimulation, 53% of the cellsremained at the G2/M stage. At 47 hrs. post-stimulation, the majority ofcells (66%) were found at the G1 stage with only a small population ofthe cells (18%) detected at G2/M. The population of the cells shiftedslightly from G1 to G2/M at 53 h post-stimulation, as 62% and 25% of thecells were detected at G1 and G2/M, respectively.

Without wishing to be bound by any theory, from these data it appearsthat agnoprotein expression prolongs the progression of cells throughoutthe cell cycle by stalling cells at the G2/M and G1 stages. This issupported by the observation that, at the earlier hours after serumstimulation (i.e., 4, 12 and 20 hrs.), the population of control andagnoprotein-producing cells which were found at each stage of the cellcycle was virtually identical to those seen at 23 h post-induction.

Example 2 Suppression of Cell Proliferation and Deregulation of the CellCycle by Agnoprotein

Stable NIH 3T3 cell lines that constitutively express agnoprotein wereproduced using pYFP-agnoprotein, which is a plasmid containing sequencesencoding the agnoprotein fused with the mutant variant of GFP called“yellow fluorescent protein” or “YFP”. Examination of YFP-agnoproteinproduction in the stably transfected NIH 3T3 cell lines showedexpression of a fusion protein which appropriately localized in thecytoplasmic perinuclear membrane of the cells. Control cells transfectedwith pEYFP-N1 (expressing YFP alone) showed no evidence for cellularcompartmentalization of YFP.

The growth rate of NIH 3T3 cell lines stably expressing eitherYFP-agnoprotein or YFP alone was examined as follows. An equal number ofeither cell type was seeded at 20,000 cells/35 mm plate in DMEM plus 10%fetal calf serum. Four independent samples were tested after 24, 48, and72 hours for cell proliferation by direct counting of the total numberof cells, and the experiments were repeated in various stable cell linesto ensure the reproducibility in various clones. FIG. 2A shows anapproximately 50% decrease in the number of agnoprotein-producing cellscompared to that seen in the control cells.

Results from FACS analysis of YFP-producing (control) NIH 3T3 cellsrevealed that these cells progress abnormally through the cell cycle. At0 hrs. post serum stimulation, greater than 90% of the YFP-producingcells were found at the G1 phase (FIG. 2B). As time progressed, thecells began to enter the S phase and accumulated at G2/M. By 21 hourspost-serum stimulation, only 39% of the cells remained at the G1 andgreater than 49% of the cells were found at G2/M. At 25 hrs.post-induction, the cells again appeared to depart from G2/M andaccumulate at the G1 phase. At 29 hrs. post-stimulation, nearly 61% ofcells were found at the G1 and only 23% were detected at G2/M.

As shown in FIG. 2C, the agnoprotein-producing NIH 3T3 cells exhibited adifferent pattern of progression through the cell cycle than the controlcells. More agnoprotein-producing cells were detected at the G2/M(39.2%) than the control (6.3%) at 0 hrs. post-serum stimulation, and50% of the agnoprotein-producing cells were found at the G1 stage atthis time point. The number of cells at the G1 stage was modestlydecreased at 16 and 21 hrs. post-stimulation, and a substantial level ofcells were detected at the G2/M phase. Further, a higher number ofagnoprotein-producing cells was found at the S phase as compared tocontrol cells. At the later times post-stimulation (i.e., 25 and 29hrs.), the number of agnoprotein-producing cells at G2/M remained athigh levels.

Without wishing to be bound by any theory, these data indicate thatexpression of agnoprotein deregulates cell cycle progression at variousstages, most noticeably by affecting cell departure from G1 to S phase,from S to G2/M, and by inducing an unusual accumulation of cells atG2/M.

Example 3 Expression of Cyclins A and B and their Kinase Activity inCells Producing Agnoprotein

The level of expression and activity of proteins involved in the controlof the cell cycle was examined in NIH 3T3 cells expressing agnoprotein.The levels of cyclin D, cdk4, cdk6, cyclin E and cdk2 levels werecomparable in synchronized NIH 3T3 cells expressing YFP (control) andNIH 3T3 cells expressing YFP-agnoprotein prepared as above. Since cyclinA/cdc2 and cyclin B/cdc2 provide the major kinase activity during G2/M,the stage which appears to be affected by agnoprotein, the effect ofagnoprotein on cyclin A and cyclin B expression was also examined.

Cyclin A—Cyclin A production was evaluated by Western blot analysis ofprotein extracts from synchronized NIH 3T3 cells expressing YFP(control) or YFP-agnoprotein prepared as above, at 0, 4, 16, 21 and 29hours after serum stimulation. Rabbit anti-cyclin A (C-19; Santa CruzBiotechnology, Inc., Santa Cruz, Calif.) was used to probe the blot.FIGS. 3A and 3B show a comparable level of cyclin A was expressed in thecontrol and agnoprotein-producing cells. The level of the “housekeeping”protein Grb2 was determined and used a s control for equal proteinloading in the gel (see lower panels of FIGS. 3A and 3B).

The associated kinase activity of the cyclin A immunocomplex wasevaluated by H1 kinase assay. FIG. 3C shows a 35% and 55% decrease incyclin A-associated kinase activity from agnoprotein-producing proteinextracts vs. control cell protein extracts at 16 and 21 hours afterserum induction, respectively. Cyclin A-associated kinase activitydeclined at 29 hrs. after serum induction in the control cells, but notin the agnoprotein producing cells (FIG. 3C).

Cyclin B—Cyclin B levels in synchronized NIH 3T3 cells expressing YFP(control) or YFP-agnoprotein were examined by Western blot analysis at0, 4, 16, 21 and 29 hrs. after serum stimulation. Mouse anti-cyclin B1(clone GNS1; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) wasused to probe the blot. Both control and agnoprotein-producing cellsshowed a noticeable increase in the level of cyclin B at 21 hrs.post-stimulation in (FIGS. 4A and 4B). However, the level of cyclin B inagnoprotein-producing cells was slightly decreased overall vs. control,particularly at 21 hrs. post-stimulation (see FIGS. 4A and 4B, lane 4).This difference was not a general event, as the levels of other cellularproteins, including cyclin A (see FIG. 3) and “housekeeping” proteinssuch as Grb2 (see lower panels of FIGS. 4A and 4B), remained unchangedin agnoprotein-producing cells relative to control cells.

The associated kinase activity of the cyclin B immunocomplex wasevaluated by H1 kinase assay. FIG. 4C shows showed a decrease in cyclinB-associated kinase activity in agnoprotein-producing cells vs. controlcells from 16 hrs. post-serum stimulation, including a 40% reduction inkinase activity at 21 hrs. post-serum stimulation.

Example 4 Expression of Tumor Suppressor Proteins in NIH 3T3 CellsExpressing Agnoprotein

The levels of cell cycle inhibitor proteins p27, p21 and p53 wereexamined by Western blot analysis on protein extracts from NIH 3T3 cellsexpressing YFP (control) or YFP-agnoprotein. The blots were probed withanti-p27 and anti-β-actin (Santa Cruz Biotechnology, Inc.); rabbitanti-p21/WAF-1 (Ab-5) and mouse anti-p53 (Ab-1) (Oncogene Science,Boston, Mass.).

FIGS. 5A showed no significant differences in the levels of p27 in thecontrol and agnoprotein-expressing cells. However, the level of p21 wasincreased in agnoprotein-producing cells as compared to control (FIG.5B). Again, the level of expression of other cellular proteins such asβ-actin remained unchanged in the control and agnoprotein-producingcells (FIG. 5C).

The ability of agnoprotein to enhance the transcriptional activity ofthe p21 promoter was also evaluated. Saos-2 cells (which lack endogenousp53) were transfected with plasmid p21-luciferase. Plasmidp21-luciferase has a p21 promoter linked to a luciferase reporter gene,and activation of the p21 promoter causes an increase in luciferaseactivity.

Saos-2 cells containing the p21-luciferase plasmid were then transfectedwith either pCMV-Agnoprotein or a plasmid expressing p53, or both. Asshown in FIG. 5D, transcriptional activity of the p21 promoter wasincreased upon expression of either agnoprotein or p53 in transfectedSaos-2 cells containing the p21-luciferase plasmid. Co-expression ofboth agnoprotein and p53 caused a synergistic elevation of p21 promoteractivity. These results indicate that agnoprotein increases the level ofp21 promoter-driven transcription, and the cooperativity of agnoproteinwith p53 can further elevate expression of p21 in cells.

Activation of p21 in agnoprotein-producing cells, which are deregulatedin G1 as well as G2/M, is noteworthy in light of recent reportsdemonstrating that p21 contributes to the regulation of the G2/Mtransition (see Niculescu A B, III et al. (1998) Mol. Cell. Biol. 18,629-643). Without wishing to be bound by any theory, the activation ofp21 in agnoprotein-producing cells may contribute to the de-regulationof G1 as well as G2/M, which leads to growth arrest.

There were also no significant differences in p53 levels of control vs.agnoprotein-producing cells (FIG. 6A). p53 is an upstream regulator ofp21. Without wishing to be bound by any theory, these data suggest thata p53 independent pathway may up-regulate expression of p21 inagnoprotein-producing cells.

Example 5 Association of Agnoprotein with p53

As p53 activity can be regulated through association with otherproteins, the ability of agnoprotein to interact with p53 was examined.Protein extracts from NIH 3T3 cells expressing YFP (control) orYFP-agnoprotein were immunoprecipitated with p53-specific antibody pAb421. Immunoprecipitated proteins were resolved by SDS-PAGE and analyzedby Western blot using a rabbit anti-Agno protein as described in DeValleL et al. (2002), supra. As shown in FIG. 6D, a distinctagnoprotein-positive band was observed for the extracts fromagnoprotein-producing cells, which was absent from the control cellextracts. These data suggest that p53 and agnoprotein are associated inthe protein extract.

To verify the interaction of agnoprotein with p53, in vitro synthesized[³⁵S]-labeled p53 was incubated with GST alone or with a GST-agnoproteinfusion protein. After 1 hr. incubation at 4° C., the complexes bound toresin were precipitated and washed with binding buffer as described inSafak M et al. (2001), supra. Bound proteins were diluted by boiling inLaemmli buffer, resolved by SDS-PAGE, and detected by autoradiography.As shown in FIG. 6B, the 53 kDa [³⁵S]-labeled p53 was retained by theGST-agnoprotein, but not by GST alone.

In the reciprocal experiment, in vitro synthesized, [³⁵S]-labeledagnoprotein was incubated with GST or GST-p53, and the bound proteinswere resolved by SDS-PAGE followed by autoradiography. As shown in FIG.6C, a band corresponding to agnoprotein was detected in GST-p53 lane,but not in the GST lane.

In a further approach to demonstrate the interaction of agnoprotein andp53, protein extracts from YFP-producing NIH-3T3 cells and controlNIH-3T3 cells expressing only YFP were mixed with GST-p53 or GST alone,and the bound proteins were analyzed by Western blot usinganti-agnoprotein antibody. As shown in FIG. 6E, a band corresponding theYFP-agnoprotein was observed in GST-p53, but not on GST alone fractions.

To determine the regions of agnoprotein which associates with p53, a GSTpull-down assay using fusion proteins in which various regions ofagnoprotein were fused to GST was conducted. The agnoprotein fusionprotein used in the pull down assay were (with numbers indicating thenumber of amino acids in the N- to C-terminal direction on the JCVagnoprotein): GST-Agno 1-71; GST-Agno 1-54; GST-Agno 1-36; GST-Agno36-71; GST-Agno 55-71; GST-Agno 18-54; and GST-Agno 18-71.

As shown in FIG. 6F, the N-terminal region of agnoprotein from aminoacids 1 to 36 is sufficient for binding to p53. This region ofagnoprotein encompasses a helix-loop-helix structure which has 63%homology with the SV40 agnoprotein.

Thus, it is evident that agnoprotein is able to interact with p53.Without wishing to be bound by any theory, the interaction ofagnoprotein and p53 may affect other events involved in the control ofcell cycle progression (such as expression of downstream targets of p53)and contribute to the inhibition of cell growth effected by agnoprotein.

All documents referred to herein are incorporated by reference. Whilethe present invention has been described in connection with thepreferred embodiments and the various figures, it is to be understoodthat other similar embodiments may be used or modifications andadditions made to the described embodiments for performing the samefunction of the present invention without deviating therefrom.Therefore, the present invention should not be limited to any singleembodiment, but rather should be construed in breadth and scope inaccordance with the recitation of the appended claims.

1. A method of inhibiting cell growth comprising introducing into a cellan effective amount of (i) an agnoprotein comprising the amino acidsequence of SEQ ID NO: 1, (ii) one or more biologically active fragmentsof agnoprotein, wherein said one or more fragments comprise amino acidresidues 1-36 of SEQ ID NO: 1, or (iii) one or more derivatives ofagnoprotein, wherein the amino acid sequence of said one or morederivatives have at least about 83% sequence identity to SEQ ID NO: 1,and wherein said one or more derivatives have cell growth inhibitoryactivity, such that growth of the cell is inhibited.
 2. The method ofclaim 1, wherein the cells are abnormally proliferating cells.
 3. Themethod of claim 2, wherein the abnormally proliferating cells are cancercells.
 4. The method of claim 2, wherein the abnormally proliferatingcells are fibroblasts.
 5. The method of claim 1 wherein the agnoproteincomprises a JCV agnoprotein.
 6. The method of claim 5, wherein the JCVagnoprotein is selected from the group consisting of SEQ ID NO: 1; SEQID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; and SEQ ID NO:
 7. 7.The method of claim 1 wherein the agnoprotein comprises a protein havingthe amino acid sequence:M-V-L-R-Q-L-S-R-K-A-S-V-K-V-S-K-T-W-S-G-T-K-K-R-A-Q-R-I-L-I-F-L-L-E-F-L-(SEQ ID NO: 13)L-D-F-C-T-G-E-D-X₁-V-D-G-K-K-R-Q-X₂-H-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-A-L-P-E-P-K-A-X₁₂,

wherein X₁ is serine or arginine; X₂ is lysine or arginine; X₃ is serineor arginine; X₄ is glycine or no amino acid; X₅ is leucine or no aminoacid; X₆ is threonine or no amino acid; X₇ is glutamine, glutamic acid,or no amino acid; X₈ is glutamine or no amino acid; X₉ is threonine,arginine, lysine or no amino acid; X₁₀ is tyrosine or no amino acid; X₁₁is serine or glycine; and X₁₂ is threonine or lysine.
 8. The method ofclaim 1 wherein the agnoprotein comprises BK virus agnoprotein or SV40agnoprotein.
 9. The method of claim 8, wherein the BK virus agnoproteinis selected from the group consisting of SEQ ID NO: 14 and SEQ ID NO:15.
 10. The method of claim 8, wherein the SV4O agnoprotein comprisesSEQ ID NO:
 17. 11. The method of claim 1, wherein the agnoproteinderivative comprises SEQ ID NO:
 22. 12. A method of treating a subjecthaving a glioblastoma, comprising administering to the subject aneffective amount of (i) an agnoprotein comprising the amino acidsequence of SEQ ID NO: 1, (ii) one or more biologically active fragmentsof agnoprotein, wherein said one or more fragments comprise amino acidresidues 1-36 of SEQ ID NO: 1, or (iii) one or more derivatives ofagnoprotein, wherein the amino acid sequence of said one or morederivatives have at least about 83% sequence identity to SEQ ID NO: 1,and wherein said one or more derivatives have cell growth inhibitoryactivity, such that growth of cells deriving from the glioblastoma isinhibited.
 13. The method of claim 12, wherein the one or moreagnoproteins, or the one or more biologically active fragments orderivatives of agnoprotein, is administered by direct injection into atissue comprising the cells deriving from a glioblastoma.
 14. The methodof claim 12, wherein the agnoprotein comprises a JCV agnoprotein. 15.The method of claim 14, wherein the JCV agnoprotein is selected from thegroup consisting of SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO:5; SEQ ID NO: 6 and SEQ ID NO:
 7. 16. The method of claim 12 wherein theagnoprotein comprises a protein having the amino acid sequence:M-V-L-R-Q-L-S-R-K-A-S-V-K-V-S-K-T-W-S-G-T-K-K-R-A-Q-R-I-L-I-F-L-L-E-F-L-(SEQ ID NO: 13)L-D-F-C-T-G-E-D-X₁-V-D-G-K-K-R-Q-X₂-H-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-A-L-P-E-P-K-A-X₁₂,

wherein X₁ is serine or arginine; X₂ is lysine or arginine; X₃ is serineor arginine; X₄ is glycine or no amino acid; X₅ is leucine or no aminoacid; X₆ is threonine or no amino acid; X₇ is glutamine, glutamic acid,or no amino acid; X₈ is glutamine or no amino acid; X₉ is threonine,arginine, lysine or no amino acid; X₁₀ is tyrosine or no amino acid; X₁₁is serine or glycine; and X₁₂ is threonine or lysine.
 17. The method ofclaim 12 wherein the agnoprotein comprises a BK virus agnoprotein orSV40 agnoprotein.
 18. The method of claim 17, wherein the BK virusagnoprotein is selected from the group consisting of SEQ ID NO: 14 andSEQ ID NO:
 15. 19. The method of claim 17, wherein the SV40 agnoproteincomprises SEQ ID NO:
 17. 20. The method of claim 12, wherein theagnoprotein derivative comprises SEQ ID NO: 22.